Applicator for hemolyzing whole blood



March 30, 1954 3 BROWN APPLICATOR FOR HEMOLYZING WHOLE BLOOD OriginalFiled Nov. 10, 1945 INVENTOR MO/PDEN G. ROWN TbRNsYs Patented Mar. 30,1954 APPLICATOR FOR HEMOLYZING' WHOLE BLOOD Moi-den G. Brown, Darien',Conn a'ssignor to American Optical Company,

Soutlibridge,

Mass a voluntary association oi Massachusetts Original applicationNovember 10, 1945, Serial No. 627,873. Divided and this applicationAugust 1, 1950, Serial N0. 177,094

1 Claim.

This application is a division of copending appliea ticn Serial No.627,873, which was filed No f fer 10, 19 -net; U. s. Patent No.2,519,997, iss'lied August 22, 1950, and which relates to a. method forconveniently, precisely and rapidly determining the hemoglobinconcentration of blood. The present application relates to apparams foruse therewith.

Various conditions of a patients blood have a direct and materialinfluence upon the health of the patient. Accordingly, accuratemeasurements of any of these conditions of the blood are highlydesirable as, for example, measurements or the hemoglobin concentrationin the blood which may be used to accurately indicate a normal conditionor an abnormal condition of the patients blood such as anemia.

Since the hemoglobin concentration or red pigmerit of the blood isdirectly associated with the patients Ieoiiditioi'i, measurements ofsaid con- A eentration are widely used by physicians for diag' noses andfor accurate records of individual case histories. Such determination ofthe concentrau e f hemoglobin in the blood is generally madecolorimetric'ally and cannot be made as aocurateiv with untreated wholeblood as with hemslyzed blood since the variations in the number ofblood cells, size and shape have a direct bearing there on. Acolorimetric determination of the hemo= globin concentration of the"blood, accordingly, may be best had when the res pigment thereof isevenly distributed throughout the blood specimen and not conoentrated incells separated by clear plas'ma. Accordingly, it is an object of thisinvention to provide improved means for subjecting a speci men of blood,which is in its most convenient form, oiryhemog'lobin, produced merelyby exposure to air, to a treatment to hemolyze the said specimen tobring about a uniform distribution of red pigment or hemoglobin therein.It is a further object of the invention to provide means to foifn thisspecimen into oxyhern'oglobin which may be formed as a layer of uniformpredeter= inined thickness whereby the light-absorbing characteristicsof this specimen may be studied and compared against a comparison memberor a series of comparison members having known light-absorbing values,to thereby obtain ah accurate indication of the hemoglobin condition oithe patients blood. I

It is also an object of the invention to provide convenient means in theform ofa rod or stick of suitable material having a handle portion anddiapered end portion for receiving a coating of 2 an hemolytic agentthereon and for supporting said coating while being applied to the bloodspecimen being hemolyzed.

Another object of the invention is to provide such a rod or stick coatedwith a blood hemolyzing agent on a tapered portion thereof was to besupported by the stick in spaced relation to a supporting surface, orthe like, and to be spaced from other-similar stick when bunchedtogether in a container for preventing adhesion between the coatingsthereon prior to their use.

Further objects and advantages of the invention will become apparentfrom a consideration of the detailed description which follows taken inconjunction with the accompanying drawings wherein a pre'ferredform ofthe apparatus and certain steps in the method to be'emplcyed therewithare shown and described. It will be understood, however, that theinvention is not to be limited to the details of the disclosure exceptas defined by the appended claim since the disclosure is given forpurposes of illustration only.

In the drawings:

Fig. 1 is a perspective view of a pair of plates which may be used withthe apparatus of the present invention, said plates being shown inspaced relation in order to disclose the details of constructionthereof;

Fig. 2 is a perspective view of the plates of Fig. 1 shown in clampedrelation in a resilient holder and ready for use;

Fig. 3 is a perspective View of the plates likewise positioned in theholder, but having a specimen-receiving area of the lower plate offsetor moved outwardly to a position for receiving-a specimen thereon, andwith 2. treated stick or rod in position for stirring the specimen onthe specimen-receiving plate;

Fig. 4 shows an enlarged view of the treated stick of the invention; I

Fig. 5 shows a carrying case or container for receiving a plurality ofsaid sticks or rods, said container forming a substantially moistureproof enclosure therefor; and

Fig. 6 is an enlarged fragmentary view showing two similarspecimen-receiving plates placed to-' gether for use in forming a bloodchamber of double thickness.

Referring to the drawings in detail, and par ticularly Fig. 1, thenumeral ill indicates generally a lower specimen-receiving plate whichmay be used in'combination with a cover plate M of equal size, generalshape and thickness. Both of said plates are transparent. The upper andlower surfaces i4 and it of the upper plate are accurately finished toprovide optically flat surfaces or parallel planes thereon. The lowerplate In is likewise finished upon it lower surface l8 and has its upperlaterally spaced side rail portions 29 and 22 accurately formed so as tobe parallel to and equally spaced from the bottom surface l8. Thesurfaces 20 and 22 are laterally spaced from a pair of intermediatespecimenreceiving areas 2 3 and 25 by means of grooves 26. and said areaare also spaced from each other by an intermediate groove 28. Thegrooves 26 and 28 are of a depth sufficient to form spaces for receivingexcess blood from the area 24 or 25 when the plates 10 and I2 areassembled one upon the other. The areas 24 and 25 for receiving theblood specimen til, as indicated in Fig. 3, are accurately formed so asto be disposed a predetermined distance below the plane of the side railportions 20 and 22, and are arranged parallel to said plane, therebyforming a space of uniform predetermined thickness between each of theseareas and the cover plate l2 when the cover plate is positionedthereover.

The plates l and I2 may be conveniently and firmly held in an assembledposition by a clamp or holder 32 which is formed of resilient material,such as bronze, stainless steel or the like, and has a base portion 34to which a handle 36 of suitable size and shape may be riveted orotherwise secured. The holder is provided with a pair of spacedresilient side members 38 extending in substantially parallel relationfrom the base portion 34, and one of said side members is provided withan upper pair of arms 40 and the other of said side members is providedwith a lower pair of arms 40' arranged in laterally spaced relation toprovide upper and lower unobstructed areas or spaces 42 aligned withspecimen-receiving areas 24 and 25 through which the specimen 38 may beobserved. The distance between the members 38 is somewhat greater thanthe combined thickness of the plates Ill and [2, as is clearly indicatedin Fig. 2, so that the lower pair of arms 40' may be provided withinwardly curved end portions 44, as shown, to engage the outer surfaceof one of the plates and press both plates into firm engagement with theother pair of resilient arms 40. Thus, when these plates are in anassembled position in the holder, they will be firmly clamped togetherand provide between the area 24 or 25 and the cover plate a space ofpredetermined thickness. A pair of indentations 65 are pressed into thebase portion 34 near opposite ends thereof so that they will be engagedby the inner edges of the plates GE! and [2 when in the holder 32 andspace these plates from the inner wall or surface of the base portion 34thus preventing any blood which may be on these edges from touching andadhering to said inner surface.

Fig. 3 shows the specimen plate and cover plate 12 in an assembledposition clamped between the resilient arms 46, 40' of the holder 32 andthe specimen plate it is positioned so that the specimen-receiving area24 is exposed and may have placed thereon a blood specimen 3!). Atreated rod or stick 45 is shown in contact with the area 24 and thespecimen 36 thereon for a purpose to be presently described. The rod orstick at is best shown in Fig. 4 and may be formed of a suitable stiffor rigid inert material, such as wood. It comprises an elongated handleportion 48 provided with straight parallel sides which extend thegreater portion of the length of the stick and a tapered portion 50adjacent an end 4 thereof as indicated at 5| so as to provide a reducedend portion for receiving an hemolytic agent 52. By the words inertmaterial as used above and hereinafter in the specification and claimsis meant a material which will not be materially affected by thehemolytic agent forming the coating 52 and which will not alter thecondition of the blood specimen under examination.

The rod or stick may be made of any preferred cross-sectional shapedesired, but as shown is circular. While the proportions or dimensionsof this rod or stick may vary somewhat, a preferred size would bebetween 1% and 1% inches in length and from to of an inch in thickness.The tapered end portions 50 of the rod or stick 46 are formed so as toreceive the coating 52 upon the outer end thereof and support saidcoating in a space entirely within the planes containing the parallelsides of the stick, whereby the hemolytic material will be normallspaced from or above any supporting surface upon which the stick rests,or spaced from the side Walls of a container or the like used therewith,or spaced from other similar sticks when bunched together in saidcontainer.

A suitable hemolytic agent which has been found to give satisfactoryresult for forming the layer or coating 52 may comprise saponin whichmay be applied to the stick by forming a tacky solution of saponin inwater or other suitable solvent and dipping the ends of the rods orsticks 46 into said solution. The coating on the rod or stick is thenair dried or dried in an oven at temperatures not exceeding 200 F.Improved results may be obtained by adding a small amount of sodiumoxalate or other anti-coagulant to the hemolytic agent to be applied tothe rods or sticks, which additional material will materially lessen thetendency of the blood specimen to coagulate.

It is desirous that the treated rod or stick 46 be formed of stiff,durable and inert material in order that the material forming thehemolytic agent will not be affected thereby. A convenient amount ofhemolytic agent upon the tapered end of the stick has been found to beprovided by dipping each stick to a depth of from to A of an inch intosaid hemolytic solution with or without the anti-coagulant as desired. Acontainer 54 is shown in Fig. 5, said container being formed of asubstantially moisture proof plastic material to serve as a suitablemeans for storing and transporting a plurality of sticks as part of theequipment of the invention. Relative long tapered portions are formed at56 and 58 on the two parts of this separable container in order toprovide a substantially air tight joint between the two parts, in orderthat moisture or dampness of the outside air may be excluded from thecontainer and thus not affect the condition of the hemolytic materialupon the ends of the sticks contained therein.

A preferred method of use of the apparatus described above for thedetermination of the hemoglobin content or concentration of the bloodspecimen comprises puncturing the finger or lobe of the ear of thepatient to obtain a small quantity of blood to be tested, said bloodbeing placed upon the area 24, or 25, of the plate I0, when in aseparated condition as shown by Fig. 1 or when in an assembled positionwith the plate [0 offset or projecting outwardly of the plate l2 andgripped by the resilient arms of the holder as shown by Fig. 3, and thismay be done by merely touching the area 24, or 25, to the finger or earlobe. The specimen 30 so obtained on the specimen-receiving area is thenagitated or stirred by the treated end of the stick 46. When thespecimen is subjected to the hemolyzing agent, such as sap-onin, for asufficient time during the stirring operation, such as for a periodranging from five to fifteen seconds, a hemolyzing action upon the bloodspecimen will be produced, which action brings about a rupturing of themembranes surrounding the individual red cells or corpuscles of theblood, allowing the red pigment or hemoglobin thereof to be evenlydistributed throughout the specimen and thereby producing a solution ofsubstantially uniform hemoglobin concentration.

The plate iii containing the hemolyzed solution so produced may then becovered by the cover plat 12 and pressed or slipped into place in theholder 32, as shown by Fig. 2, or may, if in the oiTset position shownin Fig. 3, be pressed inwardly to a position adjacent the cover plate i2so that, in either case, the prepared solution will be contained betweenthe lower surface Id of the plate E2 and the area 24, or 25, of theplate it as a uniform layer of predetermined thickness. The solution soprepared will tend to spread over the entire specimen-receiving area bycapillary attraction between these adjacent slightly spaced surfaces.Any excess solution at the outer edge of the plates Ill and I2 may bewiped therefrom and if there is an excess amount on the area when thecover plate is placed over the area 24, or 25, it may flow into thegrooves 25 or 28 or to the outer edges of the plates [0 and 12 as thelayer of predetermined thickness is formed.

When a layer of uniform thickness of prepared solution has been producedbetween the plates 12 and 52, these plates may be positioned in thefield of a colorimetric device or the like and the light-absorptionproperties thereof observed and compared with a standard comparisonmember or a series of standard comparison areas of known light-absorbingvalues. Thus when a comparison area of known value and of lightabsorbingproperties which are the same as the specimen has been ascertained anaccurate indication of the hemoglobin concentration of the specimen maybe determined therefrom.

In cases of extreme anemia wher the amount of red pigment or hemoglobinin the blood is relatively small the physician may employ to goodadvantage two specimen-receiving plates ill with the specimen-receivingarea thereon facing each other as shown by Fig. 6, thereby forming bloodchambers between each adjacent pair of areas of double predeterminedthickness as indi cated by the letters t and t and, accordingly, thespecimen placed therebetween will have double light-absorbing valueswhich may be, in said cases of extreme anemia, more accurately observedand measured or compared in a colorimetric device, or the like, thancould the specimen if it were only half as thick. Obviously, in such acombination of similar plates a blood chamber of single thickness t mayreadily b formed when 6 desired by merely placing the upper plateupside-down upon the lower plate since surfaces 2 1 and 25 are parallelto and equally spaced from the surface).

Thus, it will be seen that a convenient, accurate and enici-entmethodand apparatus have been shown and described for ascertaining thehemoglobin content or concentration of a patients blood.

Having described the invention, I claim:

A preformed applicator for use in hemolyzing a minute specimen offreshly extracted whole blood which is to be tested for determining thehemoglobin concentration thereof, said applicator comprising anelongated rod of rigid and durable material provided with a handleportion having approximately straight parallel sides which extend thegreater portion of the length of the rod and terminate in a relativelyshort portion tapered toward the end thereof and comprising theremainder of said rod, said tapered portion being coated with a thindried adherent layer of hemolytic agent comprising saponin which extendsfrom said end of the rod partially along the length of the taperedportion and being sufiiciently thin that the hemolytic agent liessubstantially entirely within the planes containing the parallel sidesof the rod whereby a plurality of such applicators may be grouped withtheir handle portions in contiguous side-by-side relation with eachother while maintaining the hemolytic agent on said tapered portionsspaced from each other, said tapered portion, at least, of the rod beingformed of a material which will not be materially affected by and whichwill not materially affect the hemolytic agent.

MORDEN G. BROWN.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 174,619 Clark Mar. 14, 1876 410,794 Hellwig Sept. 10, 1889458,244 Tyree Aug. 25, 1891 1,498,073 Cohn June 1'7, 1924 1,969,874Butterfield Aug. 14, 1934 2,008,206 Grant July 16, 1935 2,206,282Jacobius July 2, 1940 FOREIGN PATENTS Number Country Date 496,345 GreatBritain Nov. 29, 1938 OTHER REFERENCES Wintrobe, Clinical Hematology,2nd ed.,

1946, pages 127, 241, 274, 275. Lea and Febiger, Philadelphia,publishers.

Suffer, L. J., Medicine, vol. 14, 1935, page 185.

Heller et al., Jour. Lab. and Clin. Med, vol. 19, 1934, page 777.

.Todd et al., Clinical Diagnosis by Laboratory Methods, 9th ed., 1939,pages 248-251, 320, 321, 347; W. B. Saunders 00., Philadelphia,publishers.

